In ischemic brain injury, calcium entry into the cytosol triggers mitochondrial calcium accumulation and can cause mitochondrial permeability transition. Despite the importance of this phenomenon, the components of the permeability transition pore and its regulation remain poorly understood. When extensive, permeability transition can result in swelling of mitochondria and the release of pro-apoptotic factors. However, this phenomenon is also involved in a reversible low conductance calcium release mechanism in mitochondria that can be protective in neurons challenged with increases in cytosolic calcium. Preliminary data indicates that treatment of isolated brain mitochondria with estrogen decreases the mitochondrial calcium threshold for permeability transition in a receptor-dependent manner. Estrogen receptor ? has been shown to localize to mitochondria in neurons, yet the role of estrogen and estrogen receptor ? directly in mitochondria is not fully established. My lab has found that ablation of estrogen receptor ? results in decreased brain mitochondrial calcium capacity that is insensitive to estrogen, suggesting that this receptor is directly involved in the modulation of calcium capacity and mitochondrial permeability transition. Inhibition of the well-known modulator of permeability transition, cyclophilin D, normally increases calcium capacity. Strikingly, estrogen receptor ? knockout mitochondria from brain are insensitive to this inhibition. This proposal will test the hypothesis that estrogen modulates brain mitochondrial permeability transition via estrogen receptor ? in mitochondria and that this modulation provides neuroprotection by preventing mitochondrial calcium overload. I will use biochemistry and molecular biology to understand the mechanism of the modulation of mitochondrial calcium capacity by estrogen activation of estrogen receptor ?. I will also investigate the effects of estrogen-estrogen receptor ? modulation of mitochondrial calcium capacity in a cell culture model of ischemia reperfusion injury where mitochondrial permeability transition is involved. I will determine whether estrogen prevents irreversible, detrimental mitochondrial swelling by activating a protective calcium release mechanism. These findings will provide new information about a novel regulator of the mitochondrial permeability pore, as well as reveal a new pathway by which estrogen modulates ischemic brain injury.